Human Immunodeficiency Virus (HIV) is the agent of Acquired Immunodeficiency Syndrome, which is commonly referred to by its acronym AIDS. There are two major strains of this virus, designated HIV-1 and HIV-2. The HI-virus is nowadays widely disseminated and constitutes a serious threat to health and wealth worldwide, forcing public health care systems to spend tremendous amounts of money for the diagnosis of HIV and treatment of AIDS.
One of the routes for viral spread is the transfusion of infected blood or blood products. Virtually all industrialized countries, as well as many developing countries, to date require on a mandatory basis testing of all blood donations to prevent the further spread of this virus. It is the task of all diagnostic methods in the field to diagnose the infection with HIV from blood as reliably and as soon after infection as possible.
Basically, three different modes of diagnosis are available:                (1) diagnosis of viral genomic material from blood by sensitive nucleic acid diagnostic procedures like polymerase chain reaction (PCR),        (2) the detection of viral antigens from blood, and        (3) the detection of antibodies against HIV from bodily fluids.        
During the course of an HIV infection, several diagnostically distinct and diagnostically relevant phases are known. In an early phase of infection only proteins or peptides derived from the HI virus may be found (“viraemic phase”), whereas no anti-HIV antibodies are present yet. In the subsequent phase, which is termed seroconversion, antibodies against the HIV antigens appear, while the amount of viral antigens (viral load) decreases. The majority of the antibodies formed in the early phase of the seroconversion belongs to the immunoglobulin class M (IgM). Later on the immune response against HIV switches to the immunoglobulin class G (IgG), which then builds up the majority of the antibodies directed against HIV. During the further course of infection the level of anti-HIV antibodies may decrease whereas the viral load (the presence of viral particles or viral antigens) in bodily fluids may increase again. The screening for the presence of HIV infection is preferably done with serological assays detecting antibodies against HIV antigens, sometimes combined with the detection of HIV antigen. Since the immune response within a patient changes during the course of infection and also varies from patient to patient, it is important to have extremely sensitive and reliable immunoassays detecting anti-HIV antibodies belonging to the subclasses IgM and IgG. Many different approaches for the detection of HIV infections have been described. Early, reliable and sensitive detection of antibodies against viral proteins is crucial and of major importance.
Viral proteins, which often are termed viral antigens, may be only detectable at the onset of infection and in a very late stage of the disease. Assays for detection of viral antigens, like the assays measuring p24 (from HIV-1) or p26 (from HIV-2), both of which are viral core proteins, can therefore be used only in combination with other diagnostic means to reliably detect an HIV infection.
Three groups of viral antigens are theoretically available, which may induce antibody formation in the host and thus be used as antigens in diagnostic procedures. These are the envelope proteins (encoded by the env gene region), viral enzymes or regulatory proteins such as the reverse transcriptase or integrase (encoded by the pol gene region) and structural core proteins (encoded by the gag gene region). The viral envelope proteins both in HIV-1 and HIV-2 are glycoproteins that are synthesized as polypeptide precursor proteins (gp160 for HIV and gp140 for HIV-2). These high molecular weight precursors, after synthesis, are cleaved to result in gp120 and gp41 (HIV-1) or gp110 and gp36 (HIV-2), respectively. The larger polypeptides (gp120 or gp110, respectively) form a surface subunit that is associated to the membrane spanning smaller polypeptides (gp41 and gp36, respectively) via loose contacts. In many hosts (patients), envelope glycoproteins are preferred targets of the anti-viral immune response. Ratner, L., et al., Nature 313 (1985) 277-84 have demonstrated that especially the membrane spanning of these envelope proteins, i.e., gp41 or gp36, respectively, bear the most immunogenic potential among these viral proteins.
Immunoassay methods, such as, e.g., ELISA (enzyme-linked immunosorbent assay), employing polypeptides encoded by the HI virus, have been extensively used in diagnosis and screening. The viral polypeptides are either directly prepared from viral material, or are derived from in vitro or in vivo expression systems using recombinant DNA technology. Both ways of antigen production suffer from severe limitations. Polypeptides derived from viral preparations may be contaminated by viable virus or infectious genetic material, thus posing a hazard to personnel using the material. Recombinant-derived material may be contaminated by non-HIV host proteins, which may result in reduced specificity or reduced sensitivity of such assays.
In the detection of antibodies against pathogenic agents, such as viral pathogens, very frequently and to great advantage antibody detection systems according to the double antigen bridge format, e.g., described in U.S. Pat. No. 4,945,042, are used. The immunoassays according to this bridge concept require the use of an antigen directly or indirectly bound to a solid phase and of the same or a cross-reactive readily soluble antigen that is directly or indirectly detectable. The antibodies under investigation, if present, form a bridge between the solid phase bound antigen and the labelled detection antigen. Only if the two antigens are bridged by specific antibodies a positive signal is generated.
Several attempts to use a recombinantly produced gp41 as an antigen for the detection of anti-HIV antibodies have been described. Recombinantly produced gp41, with some limitations, may be used to detect anti-HIV antibodies. Such gp41 is either used alone or in combination with other HIV antigens to measure anti-HIV antibodies. Nowadays, assays are known which independently aim at the detection of both HIV antigen and/or anti-HIV antibodies. In WO 93/21346, a “combi-test” for the simultaneous detection of gp24 antigen and antibodies to HIV-1 gp41 and HIV-2 gp36 is described. In this assay, a solid phase is used to which the recombinantly produced gp41 is directly coated.
It is also well established that the use of extraordinarily high or low pH values is one way to keep gp41 (or gp36) in solution. Recombinantly produced gp41 is known to be soluble around and below pH 3.0 or around and above pH 11.0.
Unfortunately, however, both HIV-1 gp41 and HIV-2 gp36, respectively, are essentially insoluble under physiological buffer conditions.
Immunoassays in general are performed at physiological pH. Due to their insolubility under physiological buffer conditions, retroviral surface glycoprotein antigens in many immunoassays are used directly coated onto a solid phase material. Direct coating of antigens to solid phase materials, however, is detrimental in many cases and results in disadvantages like conformational changes, molecular unfolding, change in antigenicity, instability, and in background problems (cf. Butler, J. E., et al., J. Immunol. Methods 150 (1992) 77-90).
Although it is possible to solubilize a retroviral surface glycoprotein (rsgp) by means of strongly chaotropic reagents or appropriate detergents, the material solubilized in such a manner is of limited use as a diagnostic tool.
The insolubility of retroviral surface glycoproteins at physiological buffer conditions in addition renders these proteins a very difficult target of routine (bio-)chemical procedures. The vast majority of “labeling chemistries”, i.e., the chemical procedures used for binding a label, e.g., a marker group to a polypeptide, is based on nucleophilic chemistry and thus rather restricted to a pH window from about pH 6 to about pH 8 and thus only works at more or less physiological buffer conditions. These routine procedures, e.g., as described in Aslam, M. and Dent, A., The preparation of protein-protein conjugates in “Bioconjugation” (1998) 216-363, Eds. M. Aslam and A. Dent, McMillan Reference, London, either do not work properly or are difficult to carry out at the extreme pH values (or in the presence of detergents such as SDS) required to solubilize a retroviral surface glycoprotein.
As mentioned above, immunoassays according to the bridge concept have proven advantageous in a wide variety of different assays aiming at the detection of antibodies reactive with pathogenic organisms. However, due to its insolubility, it has, e.g., not been possible to use the e-gp41 molecule (i.e. “ectodomain of glycoprotein 41”) of HIV-1 or e-gp36, respectively, in such an assay setup.
In order to compensate for the disadvantages of direct coating, a variety of assays have been designed, which instead of using the e-gp41 antigen, make use of synthetically or recombinantly produced partial sequences thereof, more or less spanning the immunodominant so-called loop region. Examples of such assays are given in the patent literature discussed below.
The loop region in the extracellular part of gp41 is the non-helical apical hairpin of the molecule linking the N-terminal helical domain to the likewise helical C-terminal domain. A significant part of antisera reactive to gp41 comprises antibodies to the apical loop motif. This disulfide bridged hairpin or loop structure thus represents an immunodominant region of gp41. One bypass to overcome the problems associated with recombinantly derived gp41 therefore is the chemical production of peptides representing partial sequences of gp41. It is important to note that gp41 or gp36, respectively, as referred to in the present invention is defined as the so-called ectodomain encompassing the loop-connected N- and C-helices but lacking the N-terminal fusion peptide and the C-terminal transmembrane segment.
Peptide fragments of a variety of HIV antigens are disclosed in the relevant patent literature (Australian Patent Application No. 597884 (57733/86), and in U.S. Pat. No. 4,735,896 and No. 4,879,212). In particular, these three specifications disclose a conserved immunodominant region of the gp41 glycoprotein, the loop region of the major envelope protein of HIV-1. An analogous immunodominant region of the gp36 protein of HIV-2 has also been synthesized. Peptides corresponding to these loop regions, which constitute the apex of the ectodomain, enable an early diagnosis of HIV-1 and HIV-2 and provide for assays with sufficient but not optimal sensitivity and good specificity. Their limitations, however, become evident with respect to detection of IgM antibodies during the first days of seroconversion in certain patients.
WO 92/22573 discloses peptides having immunological properties in common with the backbone, i.e., with an immunodominant region of the transmembrane envelope protein (e.g., gp41 or gp36) of various mammalian immunodeficiency viruses. It further confirms that this immunodominant region comprises a disulfide loop which is highly conserved in immunodeficiency virus isolates derived from different mammalian species.
EP 396 559 relates to artificial peptides bearing an amino acid sequence that corresponds to a naturally occurring amino acid sequence of a HIV. The epitopes again are derived from sequences corresponding to the loop structure of gp41 or gp36, respectively. They further have been refined to contain a disulfide bridge formed by a chemical oxidation step between the two cysteine residues of the immunodominant loop.
A quite significant percentage of antibodies as contained in anti-HIV antisera of HIV-infected patients, however, does not react with the sequence motif or its variants derived from the immunodominant loop of gp41 or gp36. Whereas these peptide antigens can be used in combination with the advantageous bridge concept, antibodies reactive with epitopes outside the loop region of HIV gp41 are not detected. Not only is the very early diagnosis of an HIV infection crucial, it is also extremely important that as many subtypes of HIV-1 and HIV-2 as possible be detected. The more epitopes, especially of the correctly folded conformational epitopes of a rsgp, are present, the less likely it is to miss an infected sample due to a false negative diagnosis.
Continuous efforts have therefore been undertaken to provide larger parts of a retroviral surface glycoprotein molecule, especially of gp41 from HIV-1, in soluble form.
The biophysical as well as the biochemical properties of gp41 have been extensively studied in past years. Lu, M., et al., Nat. Struct. Biol. 2 (1995) 1075-82) have partially elucidated the trimeric structure of gp41. Since gp41 under physiological conditions forms an insoluble aggregate, the investigations were confined to truncated versions of the ectodomain gp41.
It has recently been confirmed by NMR spectroscopy (Caffrey, M., et al., J Biol Chem 275 (2000) 19877-82) that the native trimer of gp41 forms a six helix bundle comprising three parallel N-terminal central helices to which the C-terminal helices pack in an anti-parallel orientation.
High molecular aggregates of gp41 have also been described. Such aggregates most likely form by interaction of the so-called apical loop region of gp41.
By protein design, an inhibitor of HIV-1 entry into target cells has been developed by Root, M. J., et al., Science 291 (2001) 884-8. This inhibitor comprises three stretches derived from the N-terminal helical domain from the gp41 and two stretches of the C-terminal helical domain from this molecule. However, this genetically engineered construct lacks many domains and many antigenic epitopes of the native molecule, and it especially does not contain the so-called loop motif, which is known to harbor particularly immunogenic epitopes (see above).
A tremendous need therefore still exists to provide as many retroviral surface glycoprotein epitopes as possible in a soluble form. Especially, there is a need for providing such soluble antigens comprising gp41 from HIV-1 or gp36 from HIV-2, respectively, for use in various therapeutic as well as diagnostic applications.
Chaperones, which are known as classical “folding helpers”, are polypeptides that assist the folding and maintenance of structural integrity of other proteins. They possess the ability to promote the folding of a polypeptide both in vivo and in vitro. Generally, folding helpers are subdivided into folding catalysts and chaperones. Folding catalysts accelerate the rate limiting steps in protein folding due to their catalytic function. Examples of catalysts are further described below. Chaperones are known to bind to denatured or partially denatured polypeptides and thus help to re-nature proteins. Thus, unlike folding catalysts, chaperones exert a mere binding function (Buchner, J., Faseb J 10 (1996) 10-19).
Chaperones are ubiquitous stress-induced proteins involved in protein maturation, folding, translocation and degradation (Gething, M. J. and Sambrook, J., Nature 355 (1992) 33-45). Although also present under normal growth conditions, they are abundantly induced under stress conditions. This further supports the idea that their physiological function is to cope with stress conditions.
To date, several different families of chaperones are known. All these chaperones are characterized by their ability to bind unfolded or partially unfolded proteins and have a physiological function that is linked to the correct folding of proteins or the removal of denatured or aggregated protein.
Well-characterized examples of chaperones are members of so-called heat-shock families of proteins, which are designated according to their relative molecular weight; for example, hsp100, hsp90, hsp70, and hsp60, as well as the so-called shsps (small heat-shock-proteins) as described by Buchner, J., Faseb J 10 (1996) 10-19 and by Beissinger, M. and Buchner, J., Biol. Chem. 379 (1998) 245-59.
Folding catalysts, unlike chaperones, assist folding by accelerating defined rate-limiting steps, thereby reducing the concentration of aggregation-prone folding intermediates. One class of catalysts, the protein disulfide isomerases (alternatively designated as thiol-disulfide-oxido-reductases), catalyzes the formation or the rearrangement of disulfide bonds in secretory proteins. In Gram-negative bacteria, the oxidative folding of secretory proteins in the periplasm is adjusted by a cascade of protein disulfide isomerases designated DsbA, DsbB, DsbC, and DsbD (Bardwell, J. C., Mol Microbiol 14 (1994) 199-205 and Missiakas, D., et al., Embo J 14 (1995) 3415-24).
Another important class of folding catalysts referred to as peptidyl prolyl cis/trans isomerases (PPIs) comprise different members such as CypA, PpiD (Dartigalongue, C. and Raina, S., Embo J 17 (1998) 3968-80, FkpA (Danese, P. N., et al., Genes Dev 9 (1995) 387-98), trigger factor (Crooke, E. and Wickner, W., Proc Natl Acad Sci USA 84 (1987) 5216-20 and Stoller, G., et al., Embo J 14 (1995) 4939-48), and Sly D (Hottenrott, S., et al., J Biol Chem 272 (1997) 15697-701). Amongst these, FkpA, SlyD and trigger factor have been found to be related based on sequence alignments.
The peptidyl prolyl isomerase FkpA has been localized to the periplasm of Gram-negative bacteria. It has been speculated that this chaperone is important for transport and translocation of bacterial outer membrane proteins. Ramm, K. and Pluckthun, A., J Biol Chem. 275 (2000) 17106-13) have shown that FkpA exhibits its beneficial effect on correct folding of proteins in two distinct ways. First, FkpA interacts with early folding intermediates, thus preventing their aggregation. Secondly, it has the ability to reactivate inactive protein, possibly also by binding to partially unfolded species that may exist in equilibrium with the aggregated form.
Some folding helpers comprise both a catalytically active domain as well as a chaperone (or polypeptide binding) domain. Representative examples are, e.g., trigger factor (Zarnt, T., et al., J Mol Biol 271 (1997) 827-37), Wang, C. C. and Tsou, C. L., Faseb J 7 (1993) 1515-7), SurA (Behrens et al., EMBO J. (2001) 20(1), 285-294), and DsbA (Frech, C., et al., Embo J 15 (1996) 392-98). According to our observations, the same modular structure seems to be realized in the PPIases FkpA and SlyD, respectively.
It has been demonstrated in different independent systems that an enhanced expression of chaperones may facilitate the recombinant production of a polypeptide. An example thereto can be found in WO 94/08012.
It is also known that an increased production of proteins can be achieved by using a gene construct comprising a polypeptide coding sequence as well as a chaperone sequence. This fusion concept, for example, has been shown to result in a significantly increased production of the human pro-insulin in the periplasm of Escherichia coli by using a gene construct comprising the human pro-insulin gene and DsbA (Winter, J., et al., Journal of Biotechnology 84 (2000) 175-185).
The approach to use chaperones for increased production of native-like folded polypeptides is mainly due to the binding and thus solubilizing function of chaperone proteins. After recombinant production of a fusion polypeptide comprising chaperone and target protein, the chaperones are customarily cleaved off from the resulting polypeptide to yield the desired polypeptide in pure form. In contrast, the present invention is based on the beneficial solubilizing effect of an appropriate chaperone while being associated with a retroviral surface glycoprotein.